Pneumococcal (Pn) capsular polysaccharides (PS) are T independent antigens that do not induce memory responses and are poor immunogens in infants. Conjugate vaccines, in which the Pn capsular PS is covalently linked to a protein, enhance the antibody titer made to the PS and are highly successful through pediatric immunization programs in reducing invasive infections caused by Pn. Despite the success of conjugate vaccines, multiple doses are required for protection, and they are expensive to produce reducing their utility in the developing world. The mechanism by which the conjugation of protein enhances PS specific antibody production remains poorly defined, making design of second generation conjugate vaccines empiric. One of the first immunological events that occurs when an antigen is introduced after immunization is uptake by an antigen presentation cell (APC), processing of the protein portion of the conjugate and presentation of peptides with MHC II to T cells. The T cell help produced is crucial for the enhanced immunogenicity of conjugate vaccines. We have been investigating the effect of the Pn PS that is covalently attached to the carrier protein on antigen processing of the carrier protein. Using the seven valent Pn capsular PS that is covalently linked to CRM197 (a non toxic form of diphtheria toxin) that is widely used in the United States, we have found that the PS transits the APC with the carrier protein peptide and co-localizes with MHC II on the APC surface. We have also found that the serotype of the PS greatly affects antigen processing efficiency of the carrier protein and that the most efficiently processed is the most immunogenic. These data imply that the PS affects the T cell help produced by the conjugate vaccine. Our goals are to determine the fate of the Pn PS attached to the carrier protein inside the antigen presenting cell (APC). We will determine the effect of antigen processing efficiency on carrier protein specific or glycopeptide specific T cell help, and antibody titer and function. Finally we will determine if altering the charge of the Pn PS attached to the carrier protein affects antigen processing, induction of T cell help and subsequent immunogenicity. These data will provide a rationale for second generation Pn conjugate vaccines with enhanced ability to induce protective antibodies with fewer doses. PUBLIC HEALTH RELEVANCE: Vaccines called conjugates against the serious pediatric pathogen Streptococcus pneumoniae (pneumococcus) have greatly reduced infections in children. Unfortunately four expensive doses are needed to fully protect each child making the vaccine expensive to our health care system and difficult to use in the developing world. This proposal will investigate how the immune system sees this vaccine and lay the ground work for new information to design second generation vaccines that will protect children with fewer doses.